Intended Use:This 2.5 hour ELISpot kit is developed to detect and visualize of single cells secreting human IL-1α.
Principle:Add stimulated cells or cells and stimulant to the wells and incubate at 37°C in CO2 incubator for a specified period. Secreted human IL-1alpha binds to antibody coated microtiter plate,coated with human IL-1alpha capture antibody. Cells and unbounded proteins are washed away. Streptavidin-AP or Streptavidin-HRP is added and binds to the biotinylated detection antibody. Unbound proteins are washed away. Biotinylated detection antibody is added and binds to the secreted human IL-1-alpha. Substrate Solution is added. A colored precipitate forms and appears as spots at the sites of human IL-1alpha secreting location. Each individual spot representing an individual human IL-1alpha secreting cell.
Background:IL-1α is a member of interleukin 1 family. IL-1α and IL-1β recognize the same IL-1 receptor and share a number of similar biological functions. IL-1α is predominantly a cell-associated molecule whereas IL-1β is a secreted molecule. IL-1α is synthesized primarily as a 31 kD precursor that lacks a signal peptide. Cleavage of the precursor is via the cysteine protease calpain, resulting in a 17.5 kD mature IL-1 molecule. Being active in the processed form, the IL-1 precursor is also biologically active via specific cell binding. A portion of the precursor is transported to the cell surface and associated with the cell membrane. Precursor IL-1α can be released and cleaved by extracellular proteases when the cells die, and can also be cleaved by activation of the calcium-dependent, membrane-associated calpains. Nearly all microbes and microbial products induce the production of IL-1α. Furthermore, IL-1α can be produced in monocytes and other cells in the 31 kD precursor state.
IL-1α can act on macrophages or monocytes by inducing its own synthesis as well as the production of TNF and IL-6. IL-1α induces the production of IL-2, IL-2 receptors, GM-CSF and IL-4 from activated T cells, stimulates B cell proliferation and maturation, and increases immunoglobulin synthesis. IL-1α affects NK cell activation and LAK production associated with other cytokines, and induces prostaglandin synthesis in endothelial cells and smooth muscle cells, collagenase production in synovial cells, and cartilage and calcium resorption in bones
Studies have shown a connection between IL-1α and the pathogenesis of endometriotic lesions. The increased expression of both matrix-degrading MMP-1 and its major stimulatory cytokine IL-1α in endometriotic lesions and the selective co-expression in the stroma of endometriotic foci clearly suggests the involvement of the IL-1α molecule in the pathogenic mechanisms leading to local invasion and tissue destruction. Reports also indicate that the translation of the neurotransmitter gene only occurs after receiving IL-1α stimulation. This effect was supressed by co-stimulation with IL-1 receptor antagonist. High levels of IL-1α are associated with sepsis, rheumatoid arthritis, inflammatory bowel disease, acute and chronic myelogenous leukemia, insulin-dependent diabetes mellitus, and atherosclerosis.
Kit Components:A. Microtiter Plates 1x96wells PVDF-bottom Immunospot plates pre-coated with mouse anti-human IL-1alpha monoclonal antibody. B. Positive Control 1x1vial. Supplied as a lyophilized recombinant human IL-1alpha. Reconstitute with 250 ul Cell Culture Media before use. Use in 1 hour. C. Wash Buffer (20x) 1x60ml. Add 1 volume of Wash Buffer (20x) to 19 volume of ddH2O. Use in 1 week. Stored at room temperature. D. Human IL-1alpha (Biotin). 1x11ml Biotinylated mouse anti-human IL-1alpha monoclonal antibody. Ready to use. E. Streptavidin (AP)(100x) 1x120ul. Add 1 volume of Streptavidin (AP) (100x) to 100 volumes of F. (Streptavidin (AP) Diluent) before use. Use in 1 month. Stored at 4°C
F. Streptavidin (AP) Diluent 1x11ml. Protein with buffer and preservative. Ready to use. G. BCIP/NBT Substrate Solution 1x11ml. Ready to use.
Storage and Stability:Store other components at 4ºC. Stable for at least 6 months. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
